The siderophore ferric enterobactin (FeEnt) enters Escherichia coli through the FepA protein of the outer membrane. The FeEnt transport process is a high affinity, multi-component, energy dependent reaction that is prototypic of iron uptake systems in Gram-negative bacterial pathogens. Siderophore receptors like FepA obtain iron against a concentration gradient, by a unique form of active transport in a membrane bilayer that cannot sustain an ion gradient. Their biochemistry is of fundamental interest to the understanding of ligand internalization through biological membranes. Despite a wealth of structural information about FepA and other closely related outer membrane proteins, their uptake processes remain obscure. They require energy and another cell envelope protein, TonB, for functionality, but little insight exists into their underlying biochemical activities. Structural similarities among FepA and its many protein homologs suggest that they function by a common mechanism, and the proposed research considers two prominent theories of their transport, the Ball-and-Chain and Transient Pore hypotheses. Both postulates involve conformational changes that internalize bound ligands through the outer membrane. Using fluorescence spectroscopy, we will perform experiments that differentiate between these potential mechanisms. The research focuses on one preeminent question about the transport reaction: what is the function of the N-terminal globular domain that resides within the interior of the receptor proteins? Toward this end, we will spectroscopically characterize conformational motion that occurs in FepA, especially in this domain, during transport and with regard to its TonB- and energy-dependence. These studies involve biochemical measurements of uptake in wild-type and site-directed mutants of FepA, fluorescence labeling, and spectroscopic characterizations of reaction kinetics and protein conformational motion. Thus, our experiments address the biochemistry of iron uptake, using the FeEnt-FepA system as a model of energy-dependent transport through the E. coli outer membrane. This research is also directly relevant to the relationship between iron and virulence, and the development of new strategies to thwart bacterial pathogens: the uniqueness of Gram-negative bacterial iron uptake systems makes them attractive targets for new antibiotic compounds. The research project will explain how pathogenic bacteria obtain iron. Because iron is needed for bacteria to create infections in human and animal hosts, our understanding of the process will allow us to develop new drugs that prevent iron uptake, and therefore, stop bacterial pathogenesis. We will use fluorescence methods to study these questions.